8FTN

E. coli ArnA dehydrogenase domain mutant - N492A

8FTN の概要

• エントリーDOI: 10.2210/pdb8ftn/pdb
• 分子名称: Bifunctional UDP-4-amino-4-deoxy-L-arabinose formyltransferase/UDP-glucuronic acid oxidase ArnA, SULFATE ION (3 entities in total)
• 機能のキーワード: polymyxin resistance, udp-glucuronic acid decarboxylase, oxidoreductase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 41402.97

構造登録者

Sousa, M.C.,Mitchell, M.E. (登録日: 2023-01-12, 公開日: 2023-11-22)

主引用文献

Mitchell, M.E.,Gatzeva-Topalova, P.Z.,Bargmann, A.D.,Sammakia, T.,Sousa, M.C.
Targeting the Conformational Change in ArnA Dehydrogenase for Selective Inhibition of Polymyxin Resistance.
Biochemistry, 62:2216-2227, 2023

PubMed Abstract: Polymyxins are important last resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. However, pathogens have acquired resistance to polymyxins through a pathway that modifies lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N). Inhibition of this pathway is, therefore, a desirable strategy to combat polymyxin resistance. The first pathway-specific reaction is an NAD-dependent oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) catalyzed by the dehydrogenase domain of ArnA (ArnA_DH). We present the crystal structure of serovar typhimurium ArnA in complex with UDP-GlcA showing that binding of the sugar nucleotide is sufficient to trigger a conformational change conserved in bacterial ArnA_DHs but absent in its human homologs, as confirmed by structure and sequence analysis. Ligand binding assays show that the conformational change is essential for NAD binding and catalysis. Enzyme activity and binding assays show that (i) UDP-GlcA analogs lacking the 6' carboxylic acid bind the enzyme but fail to trigger the conformational change, resulting in poor inhibition, and (ii) the uridine monophosphate moiety of the substrate provides most of the ligand binding energy. Mutation of asparagine 492 to alanine (N492A) disrupts the ability of ArnA_DH to undergo the conformational change while retaining substrate binding, suggesting that N492 is involved in sensing the 6' carboxylate in the substrate. These results identify the UDP-GlcA-induced conformational change in ArnA_DH as an essential mechanistic step in bacterial enzymes, providing a platform for selective inhibition.

PubMed: 37410993
DOI: 10.1021/acs.biochem.3c00227

実験手法

X-RAY DIFFRACTION (2.8 Å)